Mapping cytosine modifications

• US 10,081,827 B2
• Filed: 01/12/2016
• Issued: 09/25/2018
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Guan, Shengxi, Dai, Nan, Zhu, Zhenyu, Correa, Jr., Ivan R., Quimby, Aine, Borgaro, Janine
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US14/993,590
• Publication Number:
  US 20160138079A1
• Time in Patent Office: 987 Days
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12P 19/30 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Y 114/11 with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)

Abstract

Methods, compositions and kits for selectively altering and detecting modified cytosine residues are provided.

Claims

1. A method for selectively altering any modified cytosine in a nucleic acid that is hydroxymethylcytosine (hmC) or methylcytosine (mC), comprising:

(a) reacting the nucleic acid with UDP-glucosamine in the presence of a glycosyltransferase to convert hmC in the nucleic acid to glucosaminylated cytosine, and

(b) reacting the nucleic acid with an oxidizing agent and UDP-glucose to convert mC to glucosylated methylcytosine wherein the oxidizing agent is methyl cytosine oxygenase or KRuO₄.

2. The method according to claim 1, wherein the reaction with UDP-glucose is catalyzed by a glycosyltransferase.

3. The method according to claim 1, further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated nucleotides and not for glucosaminylated nucleotides.

4. The method according to claim 3, wherein the endonuclease has an amino acid sequence at least 95% identical to SEQ ID NO:

3.

5. The method according to claim 3, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of SEQ ID NOs:

3-8 and SEQ ID NO;

10.

6. The method according to claim 3, comprising:

ligating an adapter to the cleaved end of the nucleic acid.

7. The method according to claim 1, wherein the methylcytosine oxygenase is a TET enzyme.

8. The method according to claim 1, wherein the methylcytosine oxygenase is mYOX1.

9. The method according to any one of claims 1-6, 7 and 8, comprising differentiating pre-existing 5-hydroxymethylcytosine from newly formed 5-hydroxymethylcytosine in a nucleic acid where 5-methylcytosine is a precursor of 5-hydroxymethylcytosine.